Process for the production of ultrapure galacto-oligosaccharides

ABSTRACT

The present invention describes a process for preparing ultrapure (≧95%) galacto-oligosaccharides (GOS), starting from GOS at lower purities by using sequential microbiological purifications involving  Saccharomyces cerevisiae  and  Streptococcus thermophilics.

FIELD OF THE INVENTION

The present invention relates to the field of preparing high purity(≧95%) galacto-oligosaccharides (GOS), starting from lactose-derivedsyrups with low GOS content.

STATE OF THE ART

Galacto-oligosaccharides (GOS) consist of a mixture of at least di-,tri-, tetra-, penta- and hexa-saccharides, in which glucose is the freereducing-end sugar and the other sugars in the chain are galactoseslinked to each other and to glucose in varying ways depending on theenzyme used in the transglycosylation reaction starting from lactose.

Currently, interest in GOS is steadily growing because recent researchhas demonstrated the efficiency of these oligosaccharides as prebiotics:in this sense they are a mixture of non-cariogenic non-digestiblelow-calorie oligosaccharides which stimulate the development ofgastrointestinal microflora.

A further benefit of GOS is their anti-adhesive activity: theseoligosaccharides can directly inhibit infections caused by entericpathogens such as E. coli, acting as mimics of the binding sites ofpathogens which generally attack gastrointestinal epithelial cells.

Commercially available GOS are synthesized from lactose by using thetransglycosylic activity of beta-galactosidases (lactases) isolated fromvarious natural microorganisms (e.g. Aspergillus oryzae, Penicilliumexpansum and Bacillus circulans) or modified microorganisms. The GOSstructure varies according to the enzyme source. The yield of GOSproduced from natural enzymes is generally 20-45% and can be increasedby employing recombinant or modified enzymes. The most widelycommercially available GOS form contains the GOS at a concentration ofabout 50-60% by weight and also contains considerable amounts of glucose(by-product of the GOS formation reaction) and non-reacted lactose(starting material). This renders them unusable by people affected bydiabetes mellitus or lactose intolerance.

Methods are known in the literature for obtaining high purity GOS,namely those in which all the digestible sugars (lactose, glucose,galactose) are absent. Such methods involve the removal of glucose andlactose in GOS by chromatography, enzymatic oxidation or microbialfermentation. The aforesaid methods however lack large-scaleapplicability (in the case of chromatography) or present drawbacks.

In Shoaf, K. et al. Infect. Immun. 74 (12) 6920-6928, 2006, a mixturecontaining mono- and disaccharide-free GOS was made in the laboratory bypreparative TLC (hence in quantities of a few milligrams).

Splechtna, B. et al. Enzyme and Microbial Technology 29 (6) 434-440,2001 describes the removal of residual lactose by its selectiveoxidation with fungal cellobiose dehydrogenase into lactobionic acid.This enzyme is not easily available and works by coupling the lactoseoxidation with reduction of 2,6-dichloro-indophenol, being present incatalytic concentrations. The oxidized redox mediator is continuouslyregenerated by the fungal laccase-catalyzed reduction of molecularoxygen into water. Ion exchange chromatographies were used to remove thelactobionic acid.

Cheng, C.-C. et al. Biotechnol. Lett. 28 793-797, 2006 describes theremoval of residual lactose by fermentation with Kluyveromycesmarxianus: the process has good yields and leads to a high purityproduct devoid of glucose, galactose, lactose, but as well as lactose,all other disaccharides present in the GOS mixture prior to treatmentwith K. marxianus are also consumed by the microorganism. Li, Z. et al.Process Biochemistry 2008, 43(8), 896-899 describes the removal ofdigestible sugars by fermentation with Saccharomyces cerevisiae orKluyveromyces lactis immobilized on calcium alginate. The results aregood when using K. lactis, but unsatisfactory when using S. cerevisiae.

The drawback of using Kluyveromyces marxianus or lactis is that althoughboth are publicly available to the public, they are not commonlyemployed in the food industry and are therefore not commerciallyavailable at low cost in ready-to-use form.

There is an evident need for an alternative method for producing highpurity GOS, which is also applicable on an industrial scale and in whichall the digestible sugars (lactose, glucose, galactose) are eitherabsent or present in small amounts, and which utilizes easily and widelyavailable microorganisms

Definitions and Abbreviations

GOS=Galacto-oligosaccharides

SUMMARY OF THE INVENTION

The present invention overcomes the aforesaid problem by means of aprocess for preparing GOS of purity ≧95% in which the overall percentageof the digestible sugars lactose, glucose and galactose is ≦5%, startingfrom GOS mixtures of a lower purity, said process comprising afermentation step with Streptococcus thermophilus and at least onefermentation step with Saccharomyces cerevisiae, where said purity iscalculated by any analytical method able to distinguish and quantify GOSand said digestible sugars.

The process enables said digestible sugars to be selectively removed bymicrobiological purification.

Advantageously said process enables a GOS mixture to be obtained withlow contents of the digestible sugars lactose, glucose and galactose, inwhich not all the disaccharides formed in the transglycosylation areremoved by microorganisms.

Advantageously the fermentation step with S. thermophilus is found toenable lactose to be selectively removed while preserving otherdisaccharides which have HPLC retention times very close to that oflactose. This distinctive preservation of the oligosaccharide,identifiable by HPLC with a peak immediately following that of lactose,makes the product obtained by the process different from others known inthe art.

The aforesaid process uses low cost commercially availablemicroorganisms widely employed in the food industry; they can be useddirectly in lyophilized form (the form in which they are purchased)without having to activate them by preparing a pre-fermentation mixture.

The aforesaid process can be easily applied on an industrial scale, i.e.on a multi-kg scale.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—shows the HPLC trace of a starting GOS sample at 40% purity, ofexample 1, obtained from lactose by transglycosylation catalyzed by B.circulans lactase;

FIG. 2—shows the HPLC trace of a GOS sample, of example 1, afterdeglucosation with Saccharomyces cerevisiae according to the presentinvention;

FIG. 3—shows the HPLC trace of a GOS sample, of example 1, afterdelactosation with Streptococcus thermophilus according to the presentinvention;

FIG. 4—shows the HPLC trace of a GOS sample, of example 1, afterdegalactosation of the sample corresponding to FIG. 3 with Saccharomycescerevisiae according to the present invention; A: after at least 20hours of fermentation; B: after at least 40 hours of fermentation;

FIG. 5—shows the HPLC trace of a GOS sample with a purity ≧95% obtainedat completion, of example 1, according to the process of the presentinvention;

FIG. 6—shows the HPLC trace of a starting GOS sample, of example 2,namely Purimune™ (GO-P90) purchased from GTC Nutrition, Golden, Colo.,USA;

FIG. 7—shows the HPLC trace of a GOS sample with a purity ≧95%, obtainedat completion, of example 2, according to the process of the presentinvention;

FIG. 8—shows the HPLC trace of a GOS sample of example 4, obtained afterfermentation with Kluveromyces marxianus.

DETAILED DESCRIPTION OF THE INVENTION

In the process of the present invention, any GOS mixture with a purityof <95% can be used as the starting material, most conveniently GOSmixtures with a purity ≧40% being used in which the impurities consistessentially of the digestible sugars lactose, glucose and galactose.

For the purposes of the present invention, to evaluate the purity of theGOS mixtures any HPLC method capable of distinguishing and quantifyingGOS, lactose, galactose and glucose is preferably used.

The term “purity” means the area percent by HPLC corresponding to thepeaks of said GOS and digestible sugars.

GOS mixtures at 40-60% purity (or higher) are available commercially(e.g. Oligomate 55, Vivinal GOS, Purimune, Cup oligo P) or can beprepared from lactose by transglycosylation catalyzed by a suitablelactase known for the purpose, such as that isolated from Bacilluscirculans. In particular, the process of the invention is convenientwhen starting from 40-60% GOS mixtures.

Preferably the process of the invention is such that said fermentationstep with S. thermophilus is followed by a fermentation step with S.cerevisiae. S. thermophilus hydrolyses lactose then consumes the glucoseproduced and accumulates galactose which is then eliminated by S.cerevisiae. By means of the fermentation step with S. thermophilus thelactose content can be reduced to the desired levels, preferably <5% byHPLC area percent, and more preferably <3%. In case the starting GOSmixture has a glucose content <5% by HPLC area percent, then saidprocess comprises a fermentation step with S. thermophilus followed by afermentation step with S. cerevisiae. S. thermophilus hydrolyses thelactose then consumes the small amount of glucose initially present andthat which it produces during accumulation of galactose, which is thenlater eliminated by S. cerevisiae.

Thus, for example (see example 2), by starting from a commerciallyavailable GOS mixture at a purity of about approximately 80% in whichglucose is present in a quantity of 2% by HPLC area percent, the lactosecontent can be reduced by direct fermentation with S. thermophilus tothe desired levels i.e. <5% by area percent relative to the sum of thearea percents of the GOS.

In case the GOS starting mixture has a glucose content ≧5% by HPLC areapercent, the fermentation step with S. thermophilus is preceded by afermentation step with S. cerevisiae; in this case the process thereforecomprises the following 3 steps:

(a) fermentation with Saccharomyces cerevisiae;

(b) fermentation with Streptococcus thermophilus;

(c) fermentation with Saccaromyces cerevisiae.

Thus, for example (see example 1), when starting from a GOS mixture witha purity of about 40% (see FIG. 1) obtained by transglycosylation withB. circulans, fermentation (a) with S. cerevisiae enables the glucosepresent in the starting GOS mixture to be almost completely removed (seeFIG. 2.)

Fermentation (b) allows lactose to be lowered to HPLC area percents ofless than 3% (see FIG. 3).

It could be seen that when starting with GOS mixtures at 40-60% purityin which glucose is present at a purity of about 20%, if fermentation(b) with S. thermophilus is preceded by a deglucosation step such asfermentation (a) then the lactose can be lowered to below 3 area %,whereas direct fermentation with S. thermophilus even if prolonged formore than 40 hours, does not enable lactose to be lowered to the desiredlevels if undertaken without first effecting a deglucosation step (a)with S. cerevisiae.

Fermentation (c) with S. cerevisiae allows residual galactose whichformed after fermentation with S. thermophilus to be almost completelyremoved (see FIG. 4).

At the end of the process, the GOS mixture obtained has a purity of ≧95%in which glucose and galactose if not absent are present in negligiblequantities which, together with lactose, nevertheless amount to ≦5% (seeFIG. 5).

Fermentations with S. cerevisiae are preferably conducted at pH 6.5±0.5at a temperature of 35±5° C. for at least 12 hours, using 40-15 g ofdehydrated S. cerevisiae per kg of dry weight of starting GOS. The step(c) can even be conducted for time periods exceeding 40 hours and insuch case, a substantial removal of the lactic acid, formed afterfermentation (b) with S. thermophilus, is actually achieved (see FIG.4B).

Fermentation with S. thermophilus is preferably undertaken at atemperature of 40±5° C., maintaining the pH at 6.0-6.5 by addition of abase, for at least 15 hours and using 1-0.4 g of dehydrated S.thermophilus per kg of dry weight of starting GOS.

According to the process of the invention, after the fermentation stepsthe mixture is preferably subjected to further processing by means ofceramic ultrafiltration, nanofiltration, decolorization with carbon andion-exchange resin deionization.

According to the process of the invention, the fermentation steps (b)and (c) are preceded by pasteurization, preferably undertaken at 75±5°C. for at least 5 minutes.

In effect, the product obtained at the end of the aforesaid processpresents a distinctive GOS profile; during the fermentation step with S.thermophilus, lactose is mainly removed, this being evident from theHPLC traces (compare FIG. 2, with FIG. 3, and FIG. 6 with FIG. 7) and itcan also be noted that in the disaccharide region there remains acomponent with a retention time of about 13.6 minutes (indicated as GOS6in the examples) immediately after the lactose retention time (about12.9 minutes). Concomitant to the lactose removal, the disappearance ofthe component with a retention time of about 12.4 minutes (indicated asGOS5 in the examples) can be noted immediately preceding the lactoseretention time. This preservation of the GOS profile is distinctive andis distinguishable from other GOS mixtures known in the state of the artsuch as those obtained by chromatographic separation or afterpurification with Kluyveromyces genus microorganisms (see example 4 andcompare FIG. 1 with FIG. 8), these methods removing indiscriminately alldisaccharides present in the GOS mixture.

Therefore the present invention also relates to GOS mixtures obtainedfrom the process i.e. GOS mixtures with a purity ≧95% in whichgalacto-disaccharides other than lactose are present in quantities ≧1%and the overall percentage of digestible sugars lactose, glucose andgalactose is ≦5%.

The aforesaid product, obtained by the process, can be employed for theknown purposes including preparation of pharmaceutical compositions,baby formulas and food compositions, these being further aspects of thepresent invention.

The present invention may be better understood in the light of thefollowing working examples.

Experimental Part

HPLC Method

This is a HPLC method using comparison with an external standard. A HPLCinstrument is used with an isocratic pump, an autosampler, a Peltieroven for column temperature control, a refraction index detector and aTransgenomic column ICE-SEP ICE-ION 300, product code ICE-99-9850equipped with an analogous precolumn. The analyses were carried outunder the following working conditions:

Column temperature: 40° C.

Injections: 20 μl

Mobile phase: H₂SO₄ 0.015N

Flow rate: 0.4 ml/minute

Analysis time 36 minutes

Integrator: Perkin Elmer Totalchrom Workstation

Under the conditions indicated above the retention time of each productwas about 12.5 minutes for lactose, 15.0 minutes for glucose, 16.2minutes for galactose, 21.3 minutes for lactic acid and 22.2 minutes forglycerin (against the external standard).

In the sample solutions, 6 peaks of GOS product were distinguished andidentified as:

GOS 1: peak at about 9.3 minutes

GOS 2: peak at about 9.7 minutes

GOS 3: peak at about 10.3 minutes

GOS 4: peak at about 11.3 minutes

GOS 5: peak at about 12.4 minutes

GOS 6: peak at about 13.6 minutes.

In the HPLC tabulation, the terms GOS1-6 simply mean the peaksattributed to GOS, irrespective of the number of saccharide unitspresent therein.

The integration system automatically calculated the contents by theformula:

%=A _(s) ×C _(STD) ×V×100/A _(STD) ×W _(s)

In which:

A_(C)=Area of the peak in the sample solution

C_(STD)=Percentage sugar concentration in the standard solution

W_(s)=Sample weight in grams

A_(STD)=Area of the sugar peak in the standard solution

During process monitoring, the contents of the GOS peaks were notcalculated against their own standards but by using the response factorof lactose or by evaluating its purity expressed as area/total area.

Using the analytical method just described, the determination of lactosewas invalidated by the possible overlap with othergalacto-oligosaccharides.

It is important to note the strong discrepancy found between thedetermination of lactose in commercial samples using the aforedescribedanalytical method and the values provided on the certificates.

For instance, the sample VIVINAL® GOS60 Batch no. 6297770 supplied byFriesland Foods Domo had a certificated lactose content on a dry matterbasis of 17.5%, while by HPLC performed in our analytical laboratory thelactose content was 33.2%.

A similar assessment in estimating lactose was found in the Purimune™sample, Batch no. 20081217 in which the certificated overall purity ofthe GOS was 90.4%, whereas by HPLC performed in our analyticallaboratory (attached) said purity was 83.0% with the purity of lactosebeing 13.6% by area percent.

The determination of lactose was therefore overestimated by ouranalytical method, resulting in an exceeding assessment of the lactoseconcentration present in the final product.

So as not to create ambiguities in the description, in the examplespresented hereinafter and in the figures section, only the purity valuesobtained with the aforedescribed HPLC method are given, as are thevalues for the commercial products.

EXAMPLE 1 Example of Preparation Starting from Lactose

80 kg of lactose monohydrate (0.22 kmols) were suspended in 120 l ofwater and heated to 70° C. under mild agitation until the lactose wascompletely dissolved.

The solution was temperature controlled at 50° C. and the pH adjustedfrom 5.5 to 5.0 with 27 ml of 75% phosphoric acid.

266 g of lactase from Bacillus circulans were added (1500 U/g).

The reaction was monitored by HPLC and after 22 hours the formation ofGOS and the appearance of glucose and galactose were detected with aconsequent lowering of the lactose area percent purity to less than 40%.

200 kg of a 40% crude GOS solution were obtained, the HPLC trace ofwhich is shown in FIG. 1 and presented in tabular form below:

Peak Delta RT Time Component Conc. Area Response Amount Norm. Area # [%][min] Name % [uV*sec] Factor [Norm. %] [%] 1 9.093 GOS 1 0.309071 1547048.6372e+06 0.8 0.8 2 0.00 9.511 GOS 2 1.338322 669890 8.6372e+06 3.5 3.53 10.137 GOS3 4.122173 2063331 8.6372e+06 10.6  10.7 4 0.75 11.082 GOS48.925738 4467730 8.6372e+06 23.0  23.2 5 — 11.433 5.511e−95 319401.000e+100   1e−94 0.2 6 12.247 GOS5 0.414881 207666 8.6372e+06 1.1 1.17 0.22 12.666 Lattasio 15.240043 7628321 8.6372e+06 39.3  39.6 8 13.372GOS6 0.471197 235855 8.6372e+06 1.2 1.2 9 0.05 15.131 Glucosio 7.5199083601195 8.2635e+06 19.4  18.7 10 0.07 16.431 Galattosio 0.395477 1905708.3150e+06 1.0 1.0 11 — 17.629 3.856e−96 2235 1.000e+100   1e−95 0.0 120.16 21.766 LCTH 0.003749 1173 5.3962e+06 0.0 0.0 13 22.328 Glicernia0.015260 6177 6.9847e+06 0.0 0.0 38.755819 19260786 2.000e+100 100.0 100.0

Step 1—Deglucosation with S. cerevisiae

The aforestated starting mixture was acidified to pH 3.0 with 250 ml of75% phosphoric acid: under these pH conditions preservation at ambienttemperature was possible.

46 kg of 40% crude GOS solution (Batch no. 01449IN9) were diluted with140 l of water and temperature controlled at 37° C. After adjusting thesolution pH from pH 2.8 to pH 7.0 with 600 ml of 24% ammonia, 200 g oflyophilized brewer's yeast were added. The deglucosation step, monitoredby HPLC, was complete after 24 hours of vigorous agitation.

The mixture was pasteurized at 70° C. for about 5 minutes thentemperature controlled at 40° C.

FIG. 2 shows the HPLC trace of the mixture after pasteurization,presented below in tabular form.

Peak Delta RT Time Component Conc. Area Response Amount Norm. Area # [%][min] Name % [uV*sec] Factor [Norm. %] [%] 1 2.62 9.275 GOS 1 0.095989402093 8.7961e+06 1.2 1.2 2 0.16 9.694 GOS 2 0.358713 1502624 8.7961e+064.4 4.3 3 10.329 GOS3 1.074666 4501696 8.7961e+06 13.3  12.9 4 2.3611.284 GOS4 2.277114 9538665 8.7961e+06 28.2  27.4 5 0.00 12.432 GOS50.105556 442165 8.7961e+06 1.3 1.3 6 0.31 12.682 Lattasio 3.84264016096537 8.7961e+06 47.6  46.2 7 0.48 13.585 GOS6 0.127645 5346968.7961e+06 1.6 1.5 8 4.14 16.059 Glucosio 0.004367 17492 8.4113e+06 0.10.1 9 16.697 Galattosio 0.082239 329174 8.4049e+06 1.0 0.9 10 — 17.8823.713e−96 17680 1.000e+100   5e−95 0.1 11 — 18.925 1.129e−96 53781.000e+100   1e−95 0.0 12 21.431 LCTH 0.020144 51870 5.4069e+06 0.2 0.113 22.683 Glicernia 0.086023 280713 6.8522e+06 1.1 0.8 14 — 25.7131.111e−95 52902 1.000e+100   1e−94 0.2 15 — 29.315 1.650e−96 78591.000e+100   2e−95 0.0 16 — 30.666 4.041e−96 19245 1.000e+100   5e−950.1 17 — 31.229 4.144e−96 19734 1.000e+100   5e−95 0.1 18 — 33.5892.158e−94 1027592 1.000e+100   3e−93 2.9 8.075096 34848113 7.000e+100100.0  100.0

Step 2—Delactosation with S. thermophilus

2.5 g/l of yeast extract were added to the above mixture (resulting fromstep 1) and the pH was adjusted from pH 5.2 to pH 6.6 with 400 ml of 15%sodium hydroxide. The mixture was inoculated with 5 g of Streptococcusthermophilus: fermentation proceeded at pH 6.4-6.5 by adding 15% sodiumhydroxide by means of a pH-stat. The end of the reaction, monitored byHPLC, was reached after 26 hours when the lactose content in the mixturewas less than 3 area %.

The mixture was pasteurized at 70° C. for about 5 minutes thentemperature controlled at 37° C.

FIG. 3 shows the HPLC trace of the mixture after pasteurization,presented below in tabular form.

Peak Delta RT Time Component Conc. Area Response Amount Norm. Area # [%][min] Name % [uV*sec] Factor [Norm. %] [%] 1 1.42 9.257 GOS 1 0.089800306895 8.8584e+06 1.3   1.4 2 0.14 9.692 GOS 2 0.296940 10148088.8584e+06 4.2   4.7 3 10.324 GOS3 0.792435 2708190 8.8584e+06 11.1  12.4 4 1.62 11.277 GOS4 1.907072 6517527 8.8584e+06 26.7   30.0 5 —12.477 2.801e−96 10806 1.000e+100 4e−95 0.0 6 12.889 Lattasio 0.163174557655 8.8584e+06 2.3   2.6 7 — 13.268 3.809e−95 146969 1.000e+100 5e−940.7 8 0.68 13.612 GOS6 0.114462 391180 8.8584e+06 1.6   1.8 9 — 14.3741.105e−95 42639 1.000e+100 2e−94 0.2 10 4.21 16.051 Glucosio 0.00787625635 8.4366e+06 0.1   0.1 11 16.701 Galattosio 1.324742 43622758.5353e+06 18.5   20.1 12 — 17.870 1.276e−95 49224 1.000e+100 2e−94 0.213 — 18.923 6.937e−95 267628 1.000e+100 1e−93 1.2 14 — 20.163 6.679e−972577 1.000e+100 9e−96 0.0 15 — 21.459 LCTH 1.524e−95 59474 1.000e+1002e−94 0.3 16 22.127 Glicernia 2.375647 4931565 5.3807e+06 33.2   22.7 170.07 22.746 0.077039 203888 6.8599e+06 1.1   0.9 18 — 23.647 1.215e−9546875 1.000e+100 2e−94 0.2 19 — 24.491 2.779e−97 1072 1.000e+100 4e−960.0 20 — 25.801 2.805e−95 108218 1.000e+100 4e−94 0.5 7.149186 217551031.000e+101 100.0    100.0

Step 3—Degalactosation with S. cerevisiae

200 g of lyophilized brewer's yeast were added to the mixture resultingfrom step 2: after 20 hours of vigorous agitation the degalactosationstep, monitored by HPLC, was complete.

The reaction was stopped by adding 5 l of 50% sulphuric acid until pH3.0 was reached.

FIG. 4 shows the HPLC trace of the mixture after degalactosation,presented below in tabular form.

Peak Delta RT Time Component Conc. Area Response Amount Norm. Area # [%][min] Name % [uV*sec] Factor [Norm. %] [%] 1 1.57 9.270 GOS 1 0.097962358655 8.7717e+06 1.6   1.8 2 0.18 9.696 GOS 2 0.312481 11440508.7717e+06 5.1   5.8 3 10.328 GOS3 0.827617 3030055 8.7717e+06 13.5  15.4 4 1.66 11.282 GOS4 1.968015 7205257 8.7717e+06 32.0   36.7 5 11.827GOS5 0.018272 66898 8.7717e+06 0.3   0.3 6 — 12.508 3.997e−96 166831.000e+100 6e−95 0.1 7 12.894 Lattasio 0.157271 575796 8.7717e+06 2.6  2.9 8 — 13.276 3.002e−95 125300 1.000e+100 5e−94 0.6 9 0.68 13.613 GOS60.159856 563296 8.7717e+06 2.5   2.9 10 — 14.381 1.490e−95 621851.000e+100 2e−94 0.3 11 4.96 16.177 Glucosio 0.021663 75309 8.3290e+060.4   0.4 12 2.15 17.117 Galattosio 0.001645 5858 8.5330e+06 0.0   0.013 — 17.881 1.395e−95 58207 1.000e+100 2e−94 0.3 14 — 18.936 6.927e−95289128 1.000e+100 1e−93 1.5 15 — 20.187 1.127e−96 4705 1.000e+100 2e−950.0 16 — 21.466 2.285e−95 95358 1.000e+100 4e−94 0.5 17 22.147 LCTH2.528752 5590606 5.2968e+06 41.1   28.5 18 0.17 22.776 Glicernia0.061780 174711 6.7754e+06 1.0   0.9 19 — 23.668 8.130e−96 339321.000e+100 1e−94 0.2 20 — 24.907 8.769e−97 3660 1.000e+100 1e−95 0.0 21— 25.733 3.543e−95 147894 1.000e+100 6e−94 0.8 6.149314 196275431.000e+101 100.0    100.0

The mixture was clarified by removing the cells by ultrafiltration.

The low molecular weight fermentation by-products (lactic acid, glycerinetc.) were then removed by nanofiltration.

The solution was then decolorized over carbon, desalinated with a pairof ion-exchange resins, namely a strong cationic (Amberlite C-200 H⁺form, 3 l) and a weak anionic (IRA-96 free base form, 3 l), arranged inseries.

The demineralised solution was then microfiltered and concentrated undervacuum until a saccharometric concentration of 75° Brix was attained.

9 kg of a GOS mixture were obtained with a purity ≧95%; the HPLC traceof the GOS mixture obtained at the end of the process is shown in FIG.5, presented below in tabular form.

Peak Delta RT Time Component Conc. Area Response Amount Norm. Area # [%][min] Name % [uV*sec] Factor [Norm. %] [%] 1 9.228 GOS1 1.828791 4189018.5581e+06 2.7 2.6 2 9.648 GOS2 6.640325 1521025 8.5581e+06 9.7 9.6 310.274 GOS3 17.010960 3896510 8.5581e+06 24.8  24.5 4 2.13 11.222 GOS439.207543 8980833 8.5581e+06 57.1  56.6 5 — 12.351 2.085e−94 557941.000e+100   3e−94 0.4 6 0.23 12.819 Lattasio 2.769116 634291 8.5581e+064.0 4.0 7 13.511 GOS6 1.233729 282596 8.5581e+06 1.8 1.8 8 — 14.3355.725e−95 15322 1.000e+100   8e−95 0.1 9 3.83 15.869 Glucosio 0.0317587003 8.2394e+06 0.0 0.0 10 — 17.703 1.500e−95 4014 1.000e+100   2e−950.0 11 — 18.822 2.397e−94 64148 1.000e+100   3e−94 0.4 68.72222315880437 4.000e+100 100.0  100.0

From the tabulated data and the attached chromatograms of the varioussteps, it can be seen that during step 2 (fermentation with S.thermophilus) the GOS5 peak disappears, while the GOS6 peak remains moreor less unaltered.

Table 1 presents a summary of the results of the various steps inexample 1:

TABLE 1 EX 1 GOS LACTOSE GLUCOSE GALACTOSE

STEP 1 (fermentation % CONC 4 3.83 0 0.09 with S. cerevisiae) % PURITY48.3 46.3 0 1 RELATIVE % 50.5 48.4 0 1

STEP 3 (fermentation % CONC 3.38 0.16 0.02 0 with S. cerevisiae) %PURITY 62.9 2.9 0.4 0 RELATIVE % 95 4.4 0.6 0

in which: % CONC = concentration in weight/weight % % PURITY = areapercent subtended by the HPLC peak RELATIVE % = purity of GOS/Σ purityof GOS, lactose, glucose and galactose

indicates data missing or illegible when filed

EXAMPLE 2 Example of Preparation Starting from GOS at 83% (90%Commercial)

200g of Purimune GOS (GO-P90) Batch no. 20081217 were solubilized in1800 ml of water. FIG. 6 shows the chromatogram and the solution HPLC ispresented below in tabular form.

Peak Time Component Conc. Area Response Amount Area # [min] Name % [μV ·s] factor norm. % Norm. [%] 1 9.181 GOS1 0.494174 209703 8.3152e+06 5.85.8 2 9.562 GOS2 0.833452 353676 8.3152e+06 9.8 9.9 3 10.072 GOS32.055456 872234 8.3152e+06 24.3  24.3 4 10.951 GOS4 3.297243 13991878.3152e+06 59.0  39.0 5 12.044 GOS5 0.202695 86014 8.3152e+06 2.4 2.4 612.428 Lattasio 1.144282 485577 8.3152e+06 13.5  13.5 7 13.041 GOS60.142975 60672 8.3152e+06 1.7 1.7 8 14.735 Glucosio 0.155892 629507.9126e+06 1.8 1.8 9 15.993 Galattosio 0.136621 64958 7.8825e+06 1.6 1.510 28.904 7.133e−97 364 1.000e+100   8e−96 0.0 8.462789 3585334 100.0 100.0

The glucose present has an area percent of <5% hence the procedurecontinued directly with fermentation by lactic acid bacteria culture.

5 g/l of yeast extract were added and the pH adjusted from pH 4.9 to pH6.4 with 0.3 ml of 15% sodium hydroxide. Following inoculation with 75mg of Streptococcus thermophilus, the fermentation proceeded at pH6.3-6.5 by adding 15% sodium hydroxide by means of a pH-stat. The end ofthe reaction, monitored by PLC, was reached after 15 hours when thelactose content of the mixture was less than 5% by area percent relativeto the sum of the area percents of GOS.

Peak Time Component Conc. Area Response Amount Area # [min] Name % [μV ·s] factor norm. % Norm. [%] 1 9.223 GOS1 0.496176 455783 8.3252e+06 6.06.4 2 9.625 GOS2 0.886683 816247 8.3252e+06 10.7 11.4 3 10.143 GOS32.031244 1865678 8.3252e+06 24.4 26.1 4 11.041 GOS4 2.578800 23686028.3252e+06 31.0 33.1 5 12.513 Lattasio 0.282604 259569 8.3252e+06 3.43.6 6 13.124 GOS6 0.074653 68606 8.3252e+06 0.9 1.0 7 16.043 Galattosio0.574881 503527 7.9890e+06 6.9 7.0 8 21.256 LCTH 1.393852 5104765.2712e+06 16.7 11.3 8.320732 7146433 100.0 100.0

A summary of the results obtained are presented in table 2.

TABLE 2 EX 2 GOS LACTOSE GLUCOSE GALACTOSE

STEP 1 (fermentation % CONC 6.07 0.28 0 1.39 with S. thermophilus) %PURITY 78 3.6 0 7 RELATIVE % 88 4 0 7.9 Abbreviations: % CONC =concentration in weight/weight % % PURITY = area percent subtended bythe HPLC peak RELATIVE % = purity of GOS/Σ purity of GOS, lactose,glucose and galactose

indicates data missing or illegible when filed

The experiment was stopped at this stage, but it was clear that themixture could be subsequently degalactosated and purified in the manneralready described in example 1.

Direct fermentation with Streptococcus thermophilus, thus avoidingdeglucosation with S. cerevisiae which in this case was superfluous,resulted in the desired area ratio of GOS to lactose. Moreover,considering that the subsequent fermentation with S. cerevisiae was ableto remove galactose, a GOS purity of 95.6% relative to the lactose alonecan theoretically be calculated based on the area percent valuesreported in table 2.

EXAMPLE 3 Example of Preparation Starting from GOS at 43% (60%Commercial)

427 g of Vivinal Gos60 Batch no. 6297770 were solubilized in 1600 ml ofwater: the HPLC data of the solution is presented below in tabular form:

Peak Time Component Conc. Area Response Amount Area # [min] Name % [μV ·s] factor norm. % Norm. [%] 1 9.365 GOS1 0.132997 117776 8.4830e+06 1.41.4 2 9.710 GOS2 0.386673 342420 8.4830e+06 4.0 4.1 3 10.230 GOS30.978558 866565 8.4830e+06 10.2 10.3 4 11.086 GOS4 2.153239 19068098.4830e+06 22.3 22.7 5 12.198 GOS5 0.283797 251318 8.4830e+06 2.9 3.0 612.556 Lattasio 3.315665 2936200 8.4830e+06 34.4 34.9 7 13.186 GOS60.153348 135798 8.4830e+06 1.6 1.6 8 14.855 Glucosio 2.070889 17191967.9525e+06 21.5 20.4 9 16.122 Galattosio 0.160862 137854 8.2093e+06 1.71.6 9.636028 8413937 100.0 100.0

The procedure was carried out exactly as in example 1, with the firstdeglucosation step with S. cerevisiae followed by fermentation with S.thermophilus; at the end of this step (step 2 in Ex. 1) an HPLC wasobtained and the result, being absolutely comparable to that of Example1, is tabulated below:

Peak Time Component Conc. Area Response Amount Area # [min] Name % [μV ·s] factor norm. % Norm. [%] 1 9.265 GOS1 0.103310 93473 8.4830e+06 1.41.6 2 9.641 GOS2 0.382881 346426 8.4830e+06 5.3 6.1 3 10.163 GOS30.908611 822101 8.4830e+06 12.6  14.5 4 11.025 GOS4 1.991562 18019418.4830e+06 27.6  31.7 5 12.104 4.315e−96 12100 1.000e+100   7e−95 0.2 612.498 Lattasio 0.167432 151490 8.4830e+06 2.3 2.7 7 13.124 GOS60.145504 144421 8.4830e+06 2.0 2.5 8 14.766 Glucosio 0.005412 45907.9525e+06 0.1 0.1 9 16.046 Galattosio 1.003410 878581 5.2093e+06 13.9 15.4 10 21.841 LCTH 2.387452 1342275 5.2712e+06 33.1  23.6 11 21.841GIOH 0.125566 89471 6.6806e+06 1.7 1.6 7.221140 5686869 100.0  100.0

It can also be seen in this example that the GOS5 peak disappears duringfermentation with S. thermophilus, while the GOS6 peak remains more orless unaltered.

As in example 2, this experiment was stopped at this stage and themixture was degalactosated and purified in the manner already describedin example 1. Moreover, considering that the second fermentation with S.cerevisiae was able to remove galactose, a GOS purity of 95.2% relativeto the lactose alone can theoretically be calculated based on the areapercent values reported in the table for the second HPLC of thisexample.

EXAMPLE 4 Example of Preparation Starting from GOS at 40% with K.marxianus (Cheng, C.-C.; et al. Biotechnol. Lett. 28 793-797, 2006)

The strain K. marxianus ATCC 56497 was cultured on a plate containingautoclave-sterilized YPD medium with agar for yeasts, and placed in anincubator at 30° C. for 48 hours.

To prepare the pre-fermentation mixture, 100 ml of liquid YPD mediumwere prepared, autoclave-sterilized then inoculated with the coloniespicked up from the previously prepared plate. The microorganisms weregrown in a shaker at a temperature of 30° C. and shaken at 200 rpm for24 hours.

The test was carried out on the same starting solution as Example 1: 460g of a 40% crude GOS solution (Batch no. 01449IN9) diluted with 1.4 l ofwater.

After adjusting the solution pH from pH 2.8 to pH 5.4 with 15% sodiumhydroxide, it was temperature controlled at 30° C.

The entire pre-fermentation mixture was used for the inoculation. Thefermentation proceeded at pH 5.2-5.4 by adding 15% sodium hydroxide bymeans of a pH-stat. Sampling of the mixture was carried out after 48hours and shows the reaction to have reached completion, as can be seenfrom the tabulated HPLC data (see also FIG. 8) presented below:

Peak Time Component Conc. Area Response Amount Area # [min] Name % [μV ·s] factor norm. % Norm. [%] 1 9.382 GOS1 0.070000 29745 8.5106e+06 1.91.5 2 9.700 GOS2 0.355858 151213 8.5106e+06 9.7 7.6 3 10.232 GOS30.944881 401505 8.5106e+06 25.9  20.0 4 11.065 GOS4 1.989309 8453118.5106e+06 54.5  42.2 5 21.900 GIOH 0.289913 96941 6.6971e+06 7.9 4.8 633.135 9.570e−94 477834 1.000e+100   3e−92 23.9 3.649961 2002549 100.0 100.0

The reaction effectively resulted in the almost complete disappearanceof lactose, but it should be noted from the attached chromatogram (FIG.8) that peaks attributable to the galacto-oligosaccharides are absentfrom the entire disaccharide region (between 12.2 min and 13.2 min).

1. A process for producing GOS mixtures with a purity ≧95% in which theoverall percentage of the digestible sugars lactose, glucose andgalactose is ≦5%, from a starting GOS mixtures of a lower purity, saidprocess comprising a fermentation step with Streptococcus thermophilusand at least one fermentation step with Saccharomyces cerevisiae, wheresaid purity is calculated by any analytical method able to distinguishand quantify GOS and said digestible sugars.
 2. The process according toclaim 1 wherein said fermentation step with S. thermophilus is followedby a fermentation step with S. cerevisiae.
 3. The process according toclaim 2 comprising, wherein when the starting GOS mixture has a glucosecontent with a HPLC area percent of ≦5%, a fermentation step with S.thermophilus is followed by a fermentation step with S. cerevisiae. 4.The process according to claim 2 wherein when the starting GOS mixturehas a glucose content with a HPLC area percent of >5%, the fermentationstep with S. thermophilus is preceded by a the fermentation step with S.cerevisiae.
 5. A The process according to claims 1 wherein thefermentations with S. cerevisiae are conducted at pH 6.5±0.5 and at atemperature of 35±° C. for at least 12 hours using 40-15 g of dehydratedS. cerevisiae per kg of dry weight of starting GOS.
 6. The processaccording to claim 1 wherein the fermentation with S. thermophilus isconducted at a temperature of 40±5° C. maintaining the pH at 6.0-6.5 byadding a base, for at least 15 hours and using 1-0.4 g of dehydrated S.thermophilus per kg of dry weight of starting GOS.
 7. The processaccording to claim 1 wherein after the fermentation steps the GOSmixture is further processed by ceramic ultrafiltration, nanofiltration,decolorization with carbon and ion-exchange resin deionization.
 8. GOSmixtures with a purity ≧95% wherein galacto-disaccharides other thanlactose are present in quantities ≧1% and the overall percentage of thedigestible sugars lactose, glucose and galactose is ≦5%.
 9. (canceled)10. A GOS mixture according to claim 8, wherein the GOS mixturecomprises at least 31% of a galactooligosaccharide present at 11.3minutes as detected by HPLC on a Perkin Elmer Totalchrom Workstation.11. Pharmaceutical or food compositions comprising the GOS mixturesaccording to claim
 8. 12. Pharmaceutical or food compositions comprisingthe GOS mixtures according to claim 10.